Critically ill patients commonly arrive at an intensive care unit with anemia. While blood transfusions are the standard of care with the intention to increase or maintain oxygen delivery to tissues, questions have been raised about the benefit of blood transfusions, particularly in non-bleeding patients with moderate anemia. A noninvasive measure of intracellular PO2 is needed to evaluate the effect and efficacy of blood transfusions on tissue and organ oxygen sufficiency. Evidenced by the explosion of new pulse oximetry and near infrared spectroscopy (NIRS) devices on the market, optical spectroscopy is attractive for noninvasive tissue monitoring in many hospital settings. But we are not there yet - no existing clinical device has overcome the critical hurdle of accurately quantifying cellular biomarkers that are sensitive to oxygenation. We propose to develop a novel, noninvasive cell oximeter that will provide real-time measurement of myoglobin saturation from optical reflectance spectra. Intracellular PO2 will be calculated from myoglobin saturation using known myoglobin oxygen dissociation curves. Continuous intracellular PO2 measurement will allow unprecedented assessment of oxygen delivery and consumption. The specific aims of this proposal are to: 1) build a three-distance optical probe; 2) develop and validate measurement of myoglobin saturation in vitro; and 3) assess the benefit of RBC transfusion in an animal model. A new probe will be built to measure spectra from superficial, intermediate, and deep tissue regions from the surface of the rabbit hind limb. In Aim 2, Parallel Factor analysis (PARAFAC) models will be developed and tested with phantoms that model the rabbit hind limb. The phantoms will have known myoglobin and hemoglobin saturations and concentrations so that the accuracy of myoglobin saturation measurement can be objectively evaluated. Finally, in Aim 3, spectra will be acquired from rabbits subjected to hemorrhagic shock. These spectra will be used to calibrate in vivo PARAFAC models. The models will enable myoglobin saturation monitoring in real time from new spectra collected from rabbits with moderate anemia that will receive RBC transfusions. The study will answer two key questions: is tissue hypoxic during moderate anemia and if so, does RBC transfusion remedy the hypoxia? Intracellular PO2 monitoring with our new device will have broad implications in clinical practice and clinical research. We envision that intracellular PO2 monitoring will become a critical component in the care of patients with oxygen insufficiency, including those with sepsis, shock, and cardiac failure. Our long-term goal is to develop the cell oximeter for clinical use in ambulances, emergency departments, intensive care units, and operating rooms.